Derivatives of pyrimido[6, 1-A]isoquinolin-4-one

ABSTRACT

The invention provides compounds or salts thereof of the general formula (I): 
                         
wherein each of R 1  and R 2  independently represents a C 1-6  alkyl or C 2-7  acyl group; X represents OCH 2  or a group CR 3 R 4 ; wherein each of R 3  or R 4  independently represents a hydrogen atom or a C 1-3  alkyl group; R 5  represents a hydrogen atom or a C 1-3  alkyl, C 2-3  alkenyl or C 2-3  alkynyl group; R 6  represents a hydrogen atom or a C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, amino, C 1-6  alkylamino, di(C 1-6 ) alkylamino or C 2-7  acylamino group; each of R 7  and R 8  independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 2-7  acyl, C 1-6  alkylthio, C 1-6  alkoxy, C 3-6  cycloalkyl; and R 9  represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1-6  alkyl, C 2-6  alkenyl, C 2-6  alkynyl, C 2-7  acyl, C 1-6  alkylthio, C 1-6  alkoxy or C 3-6  cycloalkyl group. The compounds or salts thereof are useful for treatment of respiratory disorders such as asthma. Compounds of the invention have a longer duration of action than the known compound trequinsin (9,10-dimethoxy-3 methyl-2-mesitylimino-2, 3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one).

This application is a divisional application of U.S. Ser. No.09/964,260, filed on Sep. 26, 2001, now issued U.S. Pat. No. 6,794,391;which in turn claimed priority to PCT/GB00/01193 filed on Mar. 29, 2000,published as WO 00/58308, on Oct. 5, 2000.

The present invention relates to derivatives ofpyrimido[6,1-a]isoquinolin-4-one and their application as inhibitors ofphosphodiesterase (PDE) isoenzymes. More particularly the inventionrelates to derivatives of pyrimido[6,1-a]isoquinolin-4-one and their usein medicine for example as bronchodilators with anti-inflammatoryproperties.

In all cells where cyclic AMP (cAMP) is present as a secondarymessenger, intracellular concentrations of cAMP are regulated by the twoprocesses involved in its formation and degradation. Stimulation ofmembrane bound receptors on the external surface of the cells (e.g. byβ-adrenoceptor agonists) results in activation of adenylyl cyclase togenerate cAMP from ATP. Phosphodiesterases present in the cell serve toreduce the concentration of cAMP by hydrolysing it to adenosinemonophosphate (AMP).

In a disease such as asthma, the principal cells involved in theassociated bronchoconstriction and inflammatory processes are subject toinhibitory control by cAMP. Inhibitors of type III phosphodiesteraseraise intracellular levels of cAMP, leading to relaxation of bronchialsmooth muscle, whereas inhibitors of type IV phosphodiesterase inhibitthe release of damaging mediators from pro-inflammatory cells. Thus, inprinciple, a combined PDE III/IV inhibitor should have the desirableeffects of a β-adrenoceptor agonist plus an inhaled anti-inflammatorysteroid which are currently the mainstay of treatment in severe asthma.Moreover, a combined PDE III/IV inhibitor given by inhalation shouldachieve beneficial effects similar to a β-agonist plus inhaled steroidand should be an unusually effective treatment of asthma and otherrespiratory disorders without the undesirable glucocorticoid effects ofthe steroid such as osteoporosis and the stunting of growth.

The potential adverse effects of a PDE III/IV inhibitor (e.g., nauseaand vomiting, gastric acid secretion, cardiovascular effects such asincrease cardiac contractility, vasodilation and potentialarrhythmogenic activity) should be avoidable with a compound that isdirectly delivered to the lungs by inhalation. It is desirable that thesubstance is long acting and non-irritant

An example of a pyrimido[6,1-a]isoquinolin-4-one derivative with PDEIII/IV inhibitory activity and known to possess antihypertensivevasodilator activity is trequinsin(9,10-dimethoxy-3-methyl-2-mesitylimino-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one),which is described by De Souza et al., J. Med. Chem. 27 1470-1480 (1984)and in GB-A-1597717.

As described by De Souza et al. and in GB-A-1597717, trequinsin hasvaluable pharmacological properties, and can be administered to humansubjects suffering from, for example, respiratory disorders. However, itis unsuitable for administration by inhalation because in vitro dataindicate its persistence of action is less than desirable.

It has now been found that it is possible to design certain pyrimido[6,1-a] isoquinolin-4-one derivatives which are PDE inhibitors, whichhave a longer duration of action relative to trequinsin and other usefulproperties.

According to a first aspect of the present invention there is provided acompound of general formula I:

wherein

-   each of R¹ and R² independently represents a C₁₋₆ alkyl or C₂₋₇ acyl    group;-   R⁵ represents a hydrogen atom or a C₁₋₃ alkyl, C₂₋₃ alkenyl or C₂₋₃    alkynyl group;-   R⁶ represents a hydrogen atom or a C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆    alkynyl, amino, C₁₋₆ alkylamino, di(C₁₋₆) alkylamino or C₂₋₇    acylamino group;-   each of R⁷ and R⁸ independently represents a hydrogen or halogen    atom or a hydroxy, trifluoromethyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆    alkynyl, C₂₋₇ acyl, C₁₋₆ alkylthio, C₁₋₆ alkoxy, C₃₋₆ cycloalkyl;    and-   R⁹ represents a hydrogen or halogen atom or a hydroxy,    trifluoromethyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₇ acyl,    C₁₋₆ alkylthio, C₁₋₆ alkoxy or C₃₋₆ cycloalkyl group;-   X represents OCH₂ or a group CR³R⁴, wherein each of R³ and R⁴    independently represents a hydrogen atom or a C₁₋₃ alkyl group;-   each of R¹⁰ and R¹¹ independently represents a hydrogen atom, a C₁₋₃    alkyl, C₃₋₆ cycloalkyl or phenyl group;-   Y represents an oxygen atom or a group CHNO₂, NCN, NH or NNO₂;-   n is an integer from 2 to 4;-   or a salt thereof.

As used herein the term “halogen” or its abbreviation “halo” meansfluoro, chloro, bromo or iodo.

As used herein the term “C₁₋₆ alkyl” refers to straight chain orbranched chain alkyl groups having from one to six carbon atoms.Illustrative of such alkyl groups are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl.C₁₋₁₄ alkyl groups are preferred.

As used herein the term “C₂₋₃ alkenyl” refers to straight chain orbranched chain hydrocarbon groups having from two to three carbon atomsand having in addition one double bond, of either E or Z stereochemistrywhere applicable. This term would include for example, vinyl and1-propenyl.

As used herein the term “C₂₋₃ alkynyl” refers to straight chainhydrocarbon groups having from two to three carbon atoms and having inaddition one triple bond. This term would include for example, ethynyland 1-propynyl.

As used herein the term “C₂₋₆ alkenyl” refers to straight chain orbranched chain hydrocarbon groups having from two to six carbon atomsand having in addition one double bond, of either E or Z stereochemistrywhere applicable. This term would include for example, vinyl,1-propenyl, 1- and 2-butenyl and 2-methyl-2-propenyl. C₂₋₃ alkenylgroups are preferred.

As used herein the term “C₂₋₆ alkynyl” refers to straight chain orbranched chain hydrocarbon groups having from two to six carbon atomsand having in addition one triple bond. This term would include forexample, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl,2-pentanyl, 3-pentanyl, 4-pentanyl, 2-hexanyl, 3-hexanyl, 4-hexanyl and5-hexanyl, C₂₋₃ alkynyl groups are preferred.

As used herein the term “C₁₋₆ alkoxy” refers to straight chain orbranched chain alkoxy groups having from one to six carbon atoms.Illustrative of such alkoxy groups are methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy,neopentoxy and hexoxy. C₁₋₄ alkoxy groups are preferred.

As used herein the term “C₂₋₇ acyl” refers to straight chain or branchedchain acyl groups having from two to seven carbon atoms. Illustrative ofsuch acyl groups are acetyl, propionyl (or propiono or propanoyl),isopropionyl (or isopropiono or isopropanoyl), butyryl (or butanoyl),isobutyryl (or isobutanoyl), pentanoyl (or valeryl), hexanoyl (orcapronyl) and heptanoyl.

As used herein the term “C₂₋₇ acyloxy” refers to straight chain orbranched chain acyloxy groups having from two to seven carbon atoms.Illustrative of such acyloxy groups are acetyloxy, propionyl (orpropiono or propanoyl)oxy, isopropionyl (or isopropiono orisopropanoyl)oxy, butyryl (or butanoyl)oxy, isobutyryl (orisobutanoyl)oxy, pentanoyl (or valeryl)oxy, hexanoyl (or capronyl)oxyand heptanoyloxy. C₂₋₄ acyloxy groups are preferred.

As used herein the term “C₃₋₆ cycloalkyl” refers to an alicyclic grouphaving from three to six carbon atoms. Illustrative of such cycloalkylgroups are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.Cyclopentyl and cyclohexyl groups are preferred.

As used herein the term “C₁₋₆ alkylthio” refers to straight chain orbranched chain alkylthio groups having from one to six carbon atoms.Illustrative of such alklylthio groups are methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio,tert-butylthio, pentylthio, neopentylthio and hexylthio. C₁₋₄ alkylthiogroups are preferred.

As used herein the term “C₁₋₆ alkylamino” refers to straight chain orbranched chain alkylamino groups having from one to six carbon atoms.Illustrative of such alkylamino groups are methylamino, ethylamino,propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino,tert-butylamino, pentylamino, neopentylamino and hexylamino. C₁₋₄alkylamino groups are preferred.

As used herein, the tern “di(C₁₋₆) alkylamino” refers to straight chainor branched chain di-alkylamino groups having from one to six carbonatoms in each of the alkyl groups. Illustrative of such dialkylaminogroups are di-methylamino, di-ethylamino, di-propylamino,di-isopropylamino, di-butylamino, di-isobutylamino, di-sec-butylamino,di-tert-butylamino, di-pentylamino, di-neopentylamino and di-hexylamino.Di(C₁₋₄)alkylamino groups are preferred.

As used herein, the term “C₂₋₇ acylamino” refers to straight chain orbranched chain acylamino groups having from two to seven carbon atoms.Illustrative of such acylamino groups are acetylamino, propionyl (orpropiono or propanoyl)amino, isopropionyl (or isopropiono orisopropanoyl)amino, butyryl (or butanoyl)amino, isobutyryl (orisobutanoyl)amino, pentanoyl (or valeryl)amino, hexanoyl (orcapronyl)amino and heptanoylamino. C₂₋₄ acylamino groups are preferred.

Where there is a substituent which renders a compound basic, for examplewhen R⁶ is an amino, alkylamino or dialkylamino group, addition of anacid results in a salt. The acid may be any suitable acid, and can beorganic or inorganic.

Preferred compounds of general formula I include those in which,independently or in any compatible combination:

-   each of R¹ and R² represents a C₁₋₆ alkyl, preferably a C₁₋₄ alkyl,    group;-   R¹ and R² are the same as each other;-   each of R³ and R⁴ represents a hydrogen atom;-   R⁵ represents a hydrogen atom;-   R⁶ represents a hydrogen atom;-   each of R⁷ and R⁸ represents a C₁₋₆ alkyl, preferably methyl, ethyl    or isopropyl, group;-   R⁷ and R⁸ are the same as each other,-   R⁹ represents a hydrogen atom, a halogen atom or a methyl or acetyl    group;-   Y represents an oxygen atom or a group CHNO₂; and-   n is 2.

Exemplary compounds include:

-   1.    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pynmido[6,1-a]isoquinolin-4-one;-   2.    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)3-[N-(N′-isopropylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;-   3.    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-methyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one;-   4.    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-isopropyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one;-   5.    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′,N′-dimethyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one;-   6.    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-(N′-phenylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-2-one;-   7.    9,10-Dimethoxy-3-[2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;-   8.    9,10-Dimethoxy-3-[N-(N′-nitro)-2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;-   9.    3-[N-(N′-Cyclohexylcarbamoyl)-2-aminoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;-   10.    3-(N-Carbamoyl-2-aminoethyl)-9,10-dimethoxy-2-(2-methylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;-   11    3-(N-Carbamoyl-2-aminoethyl)-2-(2,6-diisopropylphenylimino)-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;-   12.    3-(N-Carbamoyl-4-aminobutyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;-   13.    3-[N-(N′-Cyano-N″-methyl)-2-guanidinoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino)-3,4,6,7-tetrahydro-2H-pyrimindo[6,1-a]isoquinolin-4-one.

The compound:9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoylaminoethyl)-3,4,6,7 tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one isparticularly preferred.

Compounds of general formula I may be prepared by any suitable methodknown in the art and/or by the following process, which itself formspart of the invention.

According to a second aspect of the invention, there is provided aprocess for preparing a compound of general formula I as defined above,the process comprising:

-   (a) derivatising a compound of general formula II:

wherein R¹, R², R⁵, R⁶, R⁷, R⁸, R⁹, X and n are as defined for generalformula I, with one or more compounds capable of reacting at the primaryamine group of the aminoalkyl moiety (—(CH₂)_(n)—NH₂), to form acompound of general formula I; or

-   (b) when X in general formula I represents a group CR³R⁴, wherein R³    represents a hydrogen atom, R⁴ represents a hydrogen atom or a C₁₋₃    alkyl group, and R⁵ represents a hydrogen atom or a C₁₋₃ alkyl    group, hydrogenating a compound of general formula III:

wherein R¹, R², R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, Y and n are as defined forgeneral formula I; and

-   (c) optionally converting a compound of general formula I so formed    into another compound of general formula I.

The reaction conditions of step (a) are generally such as to favour thereaction, which may be a nucleophilic displacement or addition and iscarried out in a solvent which is suitable for the particular reaction.

Compounds chosen for reacting with a compound of general formula II arecapable of reacting at the primary amine group of the alkylamino moietyin the compound of general formula II, to form a compound of generalformula I. For example:

-   when Y represents an oxygen atom and each of R¹⁰ and R¹¹ represents    a hydrogen atom, a compound of general formula II may be derivatised    with sodium cyanate;-   when Y represents an oxygen atom, R¹⁰ represents a hydrogen atom and    R¹¹ represents a C₁₋₃ alkyl, C₃₋₆ cycloalkyl or phenyl group, a    compound of general formula II may be derivatised with an isocyanate    of the general formula R¹¹NCO;-   when Y represents CHNO₂, R¹⁰ represents a hydrogen atom and R¹¹    represents a C₁₋₃ alkyl or C₃₋₆ cycloalkyl group, a compound of    general formula II may be derivatised with an N—C₁₋₃ alkyl- or    N—C₃₋₆ cycloalkyl-1-(methylthio)-2-nitroethenamine of the general    formula CH₃SC(═CHNO₂)NR¹⁰R¹¹;-   when Y represents CHNO₂, a compound of general formula II may be    reacted first with 1,1-bis(methylthio)-2-nitroethylene and the    resulting compound may then be reacted with an amine of the general    formula R¹⁰R¹¹NH, wherein R¹⁰ and R¹¹ are as defined for general    formula I;-   when Y represents NH, a compound of general formula II may be    derivatised with a compound of general formula CH₃SC(═NH)NR¹⁰R¹¹ or    a salt thereof, wherein R¹⁰ and R¹¹ are as defined for general    formula I; and-   when Y represents NCN, a compound of general formula II may be    derivatised with a compound of general formula CH₃SC(═NCN)NR¹⁰R¹¹ or    a salt thereof, wherein R¹⁰ and R¹¹ are as defined for general    formula I.

In specific cases:

-   for    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,    sodium cyanate may be chosen;-   for    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-(N′-isopropylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,    isopropylisocyanate may be chosen;-   for    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-methyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one,    N-methyl-1-(methylthio)-2-nitroethenamine may be chosen;-   for    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-isopropyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one,    1,1-bis(methylthio)-2-nitroethylene and isopropylamine maybe chosen;-   for    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′,N′-dimethyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one,    1,1-bis(methylthio)-2-nitroethylene and dimethylamine may be chosen;    and-   for    9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-(N′-phenylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-2-one,    phenylisocyanate may be chosen.

Compounds of general formula II may be prepared by reacting a compoundof general formula IV:

wherein R¹, R², R⁵, R⁶, R⁷, R⁸, R⁹ and X are as defined for generalformula I, with a compound of general formula V:LG-(CH₂)_(n)—N-PG  Vwherein n is as defined for general formula I, LG represents a leavinggroup, and PG represents a protecting group; and then removing theprotecting group.

The reaction between a compound of general formula IV and a compound ofgeneral formula V is generally carried out in suitable conditions forthe reaction, which is a nucleophilic substitution. A base such as K₂CO₃may be used in the presence of NaI and the reaction is performed in asuitable solvent such as 2-butanone.

The leaving group LG in general formula V may be any suitable leavinggroup, but is preferably a halogen atom, such as bromine. The protectinggroup PG in general formula V may be any suitable protecting group, suchas a phthaloyl group. If the reaction between a compound of generalformula IV and V is carried out in a base such as K₂CO₃, the protectinggroup should be base-stable. A suitable compound of general formula V isN-(2-bromoethyl)phthalimide:

The protecting group may then be removed by standard deprotectionprocedures. For example, hydrazine hydrate may be used. The reactionconditions are generally to favour the reaction, for example in asuitable solvent such as ethanol and/or chloroform at room temperature.

Compounds of general formula IV may be prepared by reacting a compoundof general formula VI:

wherein R¹, R², R⁵, R⁶ and X are as defined for general formula I and LGrepresents a leaving group; with a compound of general formula VII:

wherein R⁷, R⁸ and R⁹ are as defined for general formula I.

Compounds of general formula VII are substituted anilines which areeither known in the art and available from commercial sources or mayreadily be prepared by methods known per se.

The leaving group LG in compounds of general formula VI may be chlorine,a thioalkyl group, preferably thiomethyl, or an alkylsulphonyl group,preferably methylsulphonyl. Preferably it is chlorine.

The reaction conditions are generally such as to favour the reaction,which is a nucleophilic displacement which is preferably carried out ina suitable solvent such as dimethylformamide or isopropanol in thepresence of a base such as potassium carbonate. Suitable reactionconditions may be found in GB-A-1597717 and EP-A-0124893, which disclosethe preparation of related compounds.

The reaction is generally applicable for producing compounds of generalformula I where R⁶ represents a hydrogen atom or a C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, amino, C₁₋₆ alkylamino or C₂₋₇ acylamino groupand R¹ to R⁵ and R⁷ to R⁹, X, Y and n have the meanings given above.

Compounds of general formula VI where LG represents a chlorine atom maybe prepared by reacting a compound of general formula VIII or a compoundof general formula IX with phosphorous oxychloride, or by heating acompound of general formula VIII with phosphorous pentachloride:

wherein R¹, R², R⁵ and R⁶ and X are as defined for general formula I.Compounds of general formula VI where LG represents a thioalkyl groupmay be prepared from compounds of formula VIII by heating withphosphorous pentasulphide in a solvent such as dioxan or pyridine togive initially the intermediate thio derivative of VIII which, ontreatment with an alkylating agent such as an alkyl iodide eg. methyliodide, in a suitable solvent such as tetrahydrofuran or ethyl acetate,gives the thioalkyl compound. Oxidation of the thioalkyl compound with,for example, 3-chloroperbenzoic acid in a solvent such as methylenechloride, gives the alkylsulphone derivative.

Compounds of general formula VIII may be prepared by reacting a compoundof general formula IX, wherein R¹, R², R⁵ and R⁶ are as defined forgeneral formula I, with a cyclodehydrating agent such as phosphorousoxychloride, under less vigorous condition, ie lower temperatures, thanthose required to give compounds of the general formula VI where LGrepresents a chlorine atom. An alternative method has been described inNL-A-6,401,827 (Hoffmann-La Roche) which involves reacting thecarbamoylmethylene-tetrahydroisoquinoline, general formula XI (whereinR¹, R², R⁵ and X have the meanings given above) with diethyl carbonatein ethanolic sodium ethoxide:

Compounds of general formula IX may be prepared by reacting a compoundof general formula XII

-   -   XII        wherein R¹, R², R⁵ and X are as defined for general formula I,        with R⁶CH(CO₂Et)₂, wherein R⁶ is as defined for general formula        I, and a strong base such as sodium ethoxide in a hot ethanolic        solution. Alternatively, the corresponding dimethyl ester can be        employed in the presence of hot methanolic sodium methoxide.

Compounds of general formula XII may be prepared by reacting a compoundof general formula XIII:

wherein R₁, R², R⁵ and X are as defined for general formula I, with ureaby heating at 160° C. Alternatively, compounds of general formula XIIImay be reacted with potassium cyanate in the presence of acetic acid ina suitable solvent such as ethanol.

Compounds of general formula XIII are either known in the art or mayreadily be prepared by methods known per se. For example, thepreparation of 1-(3,4-dimethoxyphenethyl)barbituric acid has beendescribed by B. Lal et al. in J. Med. Chem. 27 1470-1480 (1984).

Turning to step (b), the reaction conditions of step (b) are generallyto favour the hydrogenation reaction, and the reaction is generallycarried out in a suitable solvent such as an alcohol, eg ethanol, with anoble metal catalyst such as palladium, platinum, rhodium or nickel, atroom temperature. The catalyst may be supported, for example on charcoalor alumina.

Compounds of general formula III may be prepared from a compound ofgeneral formula XIV:

wherein R¹, R² and R⁶ are as defined for general formula I, and R⁴ andR⁵ independently represent a hydrogen atom or a C₁₋₃ alkyl group. Thereactions are conducted as described above for converting a compound ofgeneral formula VIII to a compound of general formula II throughcompounds of general formula VI and general formula IV, and thepreferred reaction conditions correspond accordingly.

Compounds of general formula XIV may be prepared from compounds ofgeneral formula VIII (wherein X represents a CH₂ group and R⁵ representsa hydrogen atom or a C₁₋₃ alkyl group) by heating with a noble metalcatalyst such as palladium, platinum, rhodium or nickel at a temperatureof 300 to 350° C. The catalyst may be supported on charcoal or aluminaand the reaction carried out in an inert solvent such as an aromatichydrocarbon, eg p-cymene.

In optional step (c), a compound of general formula I may be convertedinto another compound of general formula I. For example, compounds ofgeneral formula I where R⁶ represents NH₂ may be converted intocompounds of general formula I where R⁶ represents a C₁₋₆ alkylaminogroup by standard chemistry, such as by alkylation of a protectedderivative such as an acyl or a p-toluenesulphonyl derivative followedby removal of the protecting group, such as by acid hydrolysis.Compounds of general formula I where R⁶ represents a di(C₁₋₆) alkylaminogroup may be prepared by direct alkylation of the alkylamino derivative.Compounds of general formula I wherein R⁵, R⁶, R⁷, R⁸ and/or R⁹represent a C₂₋₃ alkenyl, C₂₋₆ alkenyl, C₂₋₃ alkynyl or C₂₋₆ alkynylgroup may be hydrogenated to give the corresponding compound withsaturated bonds. The reaction conditions for the hydrogenation are asoutlined above for step (b).

According to a third aspect, the present invention provides acomposition comprising a compound of general formula I and aveterinarily or pharmaceutically acceptable carrier or diluent.Preferably the composition is a pharmaceutical composition for humanmedicine.

Compounds of the present invention are PDE inhibitors and thus possessvaluable pharmacological properties, such as bronchodilator activity asdemonstrated by the inhibition of field-stimulated contraction ofguinea-pig isolated trachea, and anti-inflammatory activity asillustrated in studies on human mononuclear cells stimulated by PHA(phytohaemagglutinin). In vitro and in vivo data indicate the compoundshave a long duration of action, as demonstrated by their persistentprotective effects against histamine induced bronchospasm in theguinea-pig when inhaled directly into the lungs as a dry powder. Theinvention therefore also relates to acute, chronic or prophylactictreatment of patients suffering from respiratory disorders including, inparticular, asthma, allergic asthma, hay fever, allergic rhinitis,bronchitis, chronic obstructive pulmonary disease (COPD), adultrespiratory distress syndrome (ARDS), and cystic fibrosis. They may alsobe used topically in skin disorders such as atopic dermatitis andpsoriasis, or in ocular inflammation or any other disease includingcerebral ischaemia or auto-immune diseases in which increasingintracellular concentrations of cAMP is considered beneficial.

One or more compounds as set out in the first aspect of the inventionmay be present in association with one or more non-toxicpharmaceutically and/or veterinarily acceptable carriers and/or diluentsand/or adjuvants and/or propellants and, if desired, other activeingredients. Suitable carriers or diluents are known in the art (egHandbook of Pharmaceutical Excipients (1994) 2^(nd) Edition, Eds. A.Wade/P J Weller, The Pharmaceutical Press, American PharmaceuticalAssociation).

Preferably, the compounds and the compositions of the present inventionare administered by inhalation, for example by aerosols or sprays whichcan disperse the pharmacological active ingredient in the form of apowder or in the form of a solution or suspension. Pharmaceuticalcompositions with powder-dispersing properties usually contain, inaddition to the active ingredient, a liquid propellant with a boilingpoint below room temperature and, if desired, adjuncts, such as liquidor solid non-ionic or anionic surfactants and/or wetting agent to form astable dispersion. Pharmaceutical compositions in which thepharmacological active ingredient is in solution contain, in addition tothis, a suitable propellant, and furthermore, if necessary, anadditional solvent and/or a stabiliser. Instead of the propellant,compressed air can also be use, it being possible for this to beproduced as required by means of a suitable compression and expansiondevice. Pharmaceutical compositions may also be delivered by breathactivated inhalation devices. Dry powder compositions are preferred foradministration by inhalation

According to a fourth aspect, the present invention provides a compoundof general formula I or a composition containing a compound of generalformula I for use in medicine.

Compounds of the present invention are useful as inhibitors ofphosphodiesterase isoenzymes. The compounds or compositions of thepresent invention may be used to prevent or treat any disease in whichthe compounds or compositions are useful, but particularly a disease inwhich raising the intracellular concentration of cAMP is desirable.Examples of diseases against which compounds are useful includerespiratory disorders including, in particular, asthma, bronchitis,chronic obstructive pulmonary disease (COPD), adult respiratory distresssyndrome (ARDS), allergic asthma, hay fever, allergic rhinitis, andcystic fibrosis. They may also be used topically in skin disorders suchas atopic dermatitis or psoriasis, ocular inflammation, or any otherdisease including cerebral ischaemia or auto-immune diseases in whichincreasing intracellular concentrations of cAMP is consideredbeneficial.

This aspect of the invention is particularly relevant to the treatmentof humans, but is also applicable to general veterinary industry, inparticular domestic animals such as dogs and cats and farm animals suchas horses, pigs, cattle, sheep, etc.

Dosage levels of the order of about 0.02 mg to about 200 mg, to be takenup to three times daily, are useful in the treatment of theabove-mentioned conditions. More particularly, a dosage range of about0.2 mg to about 20 mg, taken up to three times daily, is effective. Theparticular dosage regime will however ultimately be determined by theattending physician and will take into consideration such factors as themedication being used, age, weight, severity of symptoms and/or severityof treatment being or to be applied, method of administration of themedication, adverse reactions and/or other contraindications.

The medication according to this aspect of the invention may be given toa patient together with other active agents, which may for example be adifferent compound of the present invention, or other compounds.Examples include β₂-adrenoceptor agonists, topical glucocorticoidsteroids, xanthine derivatives, antihistamine compounds, leukotrieneantagonists, inhibitors of leukotriene synthesis and/or combinationsthereof.

According to a fifth aspect, the present invention provides the use of acompound of general formula I in the manufacture of an inhibitor of atype III/IV phosphodiesterase isoenzyme. The invention encompasses theuse of a compound of general formula I in the manufacture of abronchodilator and/or an anti-asthmatic medication and/or a medicamentfor the prevention or treatment of chronic obstructive pulmonary disease(COPD).

The invention also relates to a method for the treatment or preventionof a disease in a mammal where a phosphodiesterase isoenzyme inhibitorand/or a bronchodilator would be expected to be of benefit, which methodcomprises administering to said mammal an amount of an effective,non-toxic amount of a compound of general formula I. The inventionencompasses a method of treating or preventing asthma and/or chronicobstructive pulmonary disease (COPD) in a mammal.

Preferred features of each aspect of the invention apply to each otheraspect of the invention, mutatis mutandis.

FIG. 1, referred to in Preparations 1 to 4 below, shows the route bywhich the compounds in Preparations 1 to 4 were synthesised;

FIG. 2, referred to in Example A below, is a graph showing the effect ofDMSO on cholinergic contractile response in superfused guinea pigtrachea, wherein “n” is the number of experiments;

FIG. 3, referred to in Example A below, is a graph showing the effect of10 μM of the compound of Example 1 of the present invention oncontraction of guinea pig trachea to electrical field stimulation overtime (n=3), wherein the arrow denotes commencement of washout period;

FIG. 4, referred to in Example A below, is a graph showing the effect of10 μM of the compound of Example 9 on contraction of guinea pig tracheato electrical field stimulation over time (n=3);

FIG. 5, referred to in Example A below, is a graph showing the effect of10 μM of the compound of Example 10 on contraction of guinea pig tracheato electrical field stimulation over time (n=3);

FIG. 6, referred to in Example A below, is a graph showing the effect of10 μM of the compound of Example 11 on contraction of guinea pig tracheato electrical field stimulation over time (n=3);

FIG. 7, referred to in Example A below, is a graph showing the effect of10 μM of the compound of Example 13 on contraction of guinea pig tracheato electrical field stimulation over time (n=3);

FIG. 8, referred to in Example A below, is a graph showing the effect of10 μM of, the compound of Example 8 on contraction of guinea pig tracheato electrical field stimulation over time (n=3);

FIG. 9 referred to in Example B below, is a graph showing the effect ofthe compound of Example 1 of the present invention against proliferationof human mononuclear cells stimulated by PHA, wherein each pointrepresents the mean of six experiments, and vertical lines representstandard error of the mean.

Preparation 1: Synthesis of2-Chloro-6,7-dihydro-9,10-Dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one(shown as (1) in FIG. 1)

A mixture of 1-(3,4-dimethoxyphenyl) barbituric acid (70 g, 0.24 mol),prepared according to the method described in B. Lal et al. J. Med.Chem. 27 1470-1480 (1984), and phosphorus oxychloride (300 ml, 3.22 mol)was refluxed for 2.5 h. The excess phosphorous oxychloride was removedby distillation (20 mmHg) on warming. After cooling the residue wasslurried in dioxan (100 ml) and cautiously added to a vigorously stirredice/water solution (11). Chloroform (11) was added and the resultingmixture was basified with 30% sodium hydroxide solution. The organiclayer was separated and the aqueous phase further extracted withchloroform (2×750 ml). The combined organic extracts were washed withwater (1.51), dried over magnesium sulphate and concentrated in vacuo toleave a gummy material (90 g). This was stirred in methanol for a fewminutes, filtered and washed with methanol (200 ml); diethyl ether(2×200 ml) and dried in vacuo at 40° C. to yield the title compound as ayellow/orange solid. 47 g, 62%

(300 MHz, CDCl₃) 2.96(2H, t, C₍₇₎ H₂); 3.96(6H, s, 2×OCH₃; 4.20(2H, t,C₍₆₎ H₂); 6.61(1H, s, C₍₁₎H); 6.76(1H, s, Ar—H); 7.10(1H, s, Ar—H).

Preparation 2:9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(shown as (2) in FIG. 1)

2-Chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one,prepared according to Preparation 1, (38.5 g, 0.13 mol) and2,4,6-trimethylaniline (52.7 g, 0.39 mol) in propan-2-ol (31) wasstirred and heated at reflux, under nitrogen, for 24 h. After cooling toroom temperature, the solution was evaporated in vacuo and the residuewas purified by column chromatography on silica gel, eluting withCH₂Cl₂/MeOH, initially 98:2, changing to 96:4 once the product began toelute from the column. The title compound was obtained with a slightimpurity, just above the product on tic). Yield 34.6 g, 67%.

Preparation 3:9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-N-phthalimidoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(shown as (3) in FIG. 1)

A mixture of9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one (which was prepared according to Preparation 2)(60.0 g, 0.153 mol), potassium carbonate (191 g, 1.38 mol), sodiumiodide (137 g, 0.92 mol) and N-(2-bromoethyl)phthalimide (234 g, 0.92mol) in 2-butanone (1500-ml) was stirred and heated at reflux, undernitrogen, for 4 days. After cooling to room temperature the mixture wasfiltered and the filtrate was evaporated in vacuo. The residue wastreated with methanol (1000 ml) and the solid filtered off, washed withmethanol and recrystallised from ethyl acetate to obtain the titlecompound as a pale yellow solid in yield 40.0 g, 46%. Evaporation of themother liquor and column chromatography of the residue on silica gel(CH₂Cl₂/MeOH 95:5) provided further product 11.7 g, 13.5%.

Preparation 4:9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(shown as (4) in FIG. 1)

A mixture of9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-N-phthalimidoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(22.0 g, 0.039 mol), prepared according to Preparation 3, and hydrazinehydrate (11.3 g, 0.195 mol) in chloroform (300 ml) and ethanol (460 ml)was stirred at room temperature, under nitrogen, for 18 h. Furtherhydrazine hydrate (2.9 g, 0.05 mol) was added and the mixture wasstirred a further 4 h. After cooling in ice/water, the solid was removedby filtration and the filtrate evaporated in vacuo. The residue wasdissolved in dichloromethane and the insoluble material was removed byfiltration. The fitrate was dried (MgSO₄) and evaporated in vacuo toafford the title compound as a yellow foam in yield 16.2 g, 96%.

EXAMPLE 19,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

Sodium cyanate (6.0 g, 0.092 mol) in water (100 ml) was added dropwiseto a stirred solution of9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,prepared according to Preparation 4 above (20.0 g, 0.046 mol) in water(600 ml) and 1N HCl (92 ml) at 80° C. After stirring for 2 h at 80° C.the mixture was cooled in an ice-bath and basified with 2N NaOH. Themixture was extracted with dichloromethane (3×200 ml) and the combinedextract was dried (MgSO₄) and evaporated in vacuo. The resulting yellowfoam was purified by column chromatography on silica gel eluting withCH₂Cl₂/MeOH (97:3) and triturated with ether to obtain the titlecompound as a yellow solid, 11.9 g, 54%.

M.p.: 234-236° C. m/z: C₂₆H₃₁N₅O₄ requires M = 477 found (M + 1) = 478HPLC: Area (%) 99.50 Column ODS (150 × 4.6 mm) MP pH3 KH₂PO₄/CH₃CN(60/40) FR (ml/min) 1.0 RT (min) 9.25 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 1.92 (1H, br s, NH), 2.06 (6H, s, 2×CH₃),2.29 (3H, s, CH₃), 2.92 (2H, t, CH₂), 3.53 (2H, m, CH₂), 3.77 (3H, s,OCH₃), 3.91 (3H, s, OCH₃), 4.05 (2H, t, CH₂), 4.40 (2H, t, CH₂), 5.35(2H, br s, NH₂), 5.45 (1H, s, C═CH), 6.68 (1H, s, ArH), 6.70 (1H, s,ArH), 6.89 (2H, s, 2×ArH).

EXAMPLE 29,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-(N′-isopropylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

Isopropylisocyanate (0.15 g, 1.77 mmol) was added dropwise to a stirredsolution of of9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(prepared according to Preparation 4 above) (0.7 g, 1.61 mmol) intoluene (6 ml) at room temperature, under nitrogen. After 2 h thesolution was evaporated in vacuo and the residue was purified by columnchromatography on silica gel (CH₂Cl₂/MeOH, 97:3) and triturated withether to obtain an off-white solid, 0.42 g, 50%.

M.p.: 181-182° C. m/z: C₂₉H₃₇N₅O₄ requires M = 519 found (M + 1) = 520HPLC: Area (%) 94.99 Column ODS (150 x 4.6 mm) MP pH3 KH₂PO₄/CH₃CN(40/60) FR (ml/min) 1.0 RT (min) 7.985 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 0.89 (6H, d, 2×CH₃), 2.05 (6H, s, 2×CH₃),2.29 (3H, s, CH₃), 1.94 (1H, br s, NH), 2.90 (2H, t, CH₂), 3.49 (2H, m,CH₂), 3.77 (3H, s, OCH₃), 3.91 (3H, s, OCH₃), 4.05 (2H, t, CH₂), 4.37(2H, t, CH₂), 5.02 (1H, br s, NH), 5.46 (1H, s, C═CH), 6.67 (1H, s,ArH), 6.69 (1H, s, ArH), 6.87 (2H, s, 2×ArH).

EXAMPLE 39,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-methyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one

A mixture of9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,prepared according to Preparation 4 above (0.8 g, 1.84 mmol) andN-methyl-1-(methylthio)-2-nitroethenamine (0.30 g, 2.03 mmol) in toluene(20 ml) was stirred and heated at reflux, under nitrogen, for 2 h. Aftercooling to room temperature the solvent was evaporated in vacuo and theresidue was purified by column chromatography on silica gel(CH₂Cl₂/MeOH, 97:3). The title compound was obtained as a yellow foam inyield 0.61 g, 62%, which on triturating with ether yielded a yellowsolid 0.40 g, 41%.

M.p.: 126-130° C. m/z: C₂₈H₃₄N₆O₅ requires M = 534 found (M + 1) = 535HPLC: Area (%) 98.98 Column ODS (150 × 4.6 mm) MP pH4 KH₂PO₄/CH₃CN(45/55) FR (ml/min) 1.0 RT (min) 6.635 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 2.07 (6H, s, 2×CH₃), 2.29 (3H, s, CH₃), 2.45(3H, d, NHCH₃), 2.92 (2H, t, CH₂), 3.65 (2H, m, CH₂), 3.77 (3H, s,OCH₃), 3.90 (3H, s, OCH₃), 4.08 (2H, t, CH₂), 4.32 (2H, m, CH₂), 5.46(1H, s, ═CH), 6.51 (1H, s, CHNO₂), 6.70 (2H, s, 2×ArH), 6.90 (2H, s,2×ArH), 8.78 (1H, m, NH), 10.35 (1H, m, NH).

EXAMPLE 49,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-isopropyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one

1,1-Bis(methylthio)-2-nitroethylene (5.3 g, 32.2 mmol) and9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,prepared according to Preparation 4 above (1.4 g, 3.22 mmol) in toluene(20 ml) was stirred and heated at reflux, under nitrogen, for 2 h. Aftercooling to room temperature the solvent was evaporated in vacuo and theresidue was purified by column chromatography on silica gel(CH₂Cl₂/MeOH, 99:1). This yielded intermediate compound (shown ascompound A below) as an oil which became a light beige solid upontrituration with ether. Yield 0.95 g, 53%.

Isopropylamine (5 ml) was added to a stirred solution of (A) (0.7 g,1.27 mmol) in dichloromethane (10 ml) and heated at reflux, undernitrogen, for 20 h. After cooling, the solution was evaporated in vacuoand the residue was purified by column chromatography on silica gel(CH₂Cl₂/MeOH, 98:2). The title compound (shown as compound B below) wasobtained as a foam in yield 0.64 g, 89%. This became a pale yellow solid(0.38 g) upon trituration with ether.

M.p.: 144-146° C. m/z: C₃₀H₃₈N₆O₅ requires M = 562 found (M + 1) = 563HPLC: Area (%) 97.57 Column ODS (150 × 4.6 mm) MP pH4 KH₂PO₄/CH₃CN(40/60) FR (ml/min) 1.0 RT (min) 9.028 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 0.87 (6H, d, CH(CH₃)₂), 2.05 (6H, s, 2×CH₃),2.29 (3H, s, CH₃), 2.93 (2H, m, CH₂), 3.48 (1H, m CH(CH₃)₂), 3.68 (2H,m, CH₃), 3.78 (3H, s, OCH₃), 3.91 (3H, S, OCH₃), 4.09 (2H, t, CH₂), 4.34(2H, m, CH₂), 5.48 (1H, s, C═CH), 6.68 (1H, s, CHNO₂), 6.69 (2H, s,2×ArH), 6.90 (2H, s, 2×ArH), 7.04 (1H, d, NH), 10.75 (1H, m, NH).

EXAMPLE 59,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)3-[N-[1-(N′,N′-dimethyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]-isoquinolin-4-one

A solution of compound A shown in Example 4 above (1.0 g, 1.81 mmol) anddimethylamine (33% in EtOH, 5.0 ml, 28 mmol) in dichloromethane (10 ml)was stirred at 35° C., under nitrogen, for 18 h. The solution was thenevaporated in vacuo and the residue was purified by columnchromatography on silica gel (CH₂Cl₂/MeOH, 97:3) to obtain the titlecompound as a yellow foam in yield 0.73 g, 73%. This became a paleyellow solid (0.60 g) upon trituration with ether.

M.p.: 187-189° C. m/z: C₂₉H₃₆N₆O₅ requires M = 548 found (M + 1) = 549HPLC: Area (%) 97.89 Column ODS (150 × 4.6 mm) MP pH4 KH₂PO₄/CH₃CN(45/55) FR (ml/min) 1.0 RT (min) 6.768 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 2.02 (6H, s, 2×CH₃), 2.28 (3H, s, CH₃), 2.95(2H, m, CH), 2.95 (6H, s, N(CH₃)₂), 3.78 (3H, s, OCH₃), 3.81 (2H, t,CH₂), 3.90 (3H, s, OCH₃), 4.05 (2H, t, CH₂), 4.55 (2H, t, CH₂), 5.43(1H, s, C═CH), 6.47 (1H, s, ArH), 6.67 (1H, s, CHNO₂), 6.70 (1H, s,ArH), 6.89 (2H, s, 2×ArH), 9.35 (1H, m, NH).

EXAMPLE 69,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-(N′-phenylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-2-one

Phenylisocyanate (0.16 g, 1.38 mmol) was added dropwise to a stirredsolution of9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,prepared according to Preparation 4 above (0.6 g, 1.38 mmol) in toluene(5 ml) at room temperature, under nitrogen. After 1 h the solvent wasevaporated in vacuo and the residue was purified by columnchromatography on silica gel (CH₂Cl₂/MeOH, 95:5). After trituration withether the title compound was obtained as a pale yellow solid 0.61 g,80%.

M.p.: 116-118° C. m/z: C₃₂H₃₃N₅O₄ requires M = 553 found (M + 1) = 554HPLC: Area (%) Area (%) 98.80 Column ODS (150 × 4.6 mm) MP 0.02 MKH₂PO₄/CH₃CN (42/58) FR (ml/min) 0.8 RT (min) 10.622 Detection 254 nm

¹H NMR (300 MHz, CDCl₃): δ 2.05 (6H, s, 2×CH₃), 2.30 (3H, s, CH₃), 2.92(2H, t, CH₂), 3.67 (2H, m, CH₂), 3.78 (3H, s, OCH₃), 3.91 (3H, s, OCH₃),4.06 (2H, t, CH₂), 4.47 (2H, t, CH₂), 5.51 (1H, s, C═CH), 5.60 (1H, sbr, NH), 6.69 (1H, s, ArH), 6.72 (1H, s, ArH), 6.89 (2H, s, 2×ArH),6.90-7.23 (5H, m, 5×ArH), 7.62 (1H, br s, NH).

EXAMPLE 79,10-Dimethoxy-3-[2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

1,3-Di-(tert-butoxycarbonyl)thiourea (1)

Sodium hydride (60% in oil, 4.7 g, 0.117 mol) was washed with petroleumether to remove the oil, then added in portions to a stirred solution ofthiourea (2.0 g, 0.026 mol) in tetrahydrofuran (400 ml) at 0° C., undernitrogen. The mixture was stirred for 5 minutes at 0° C. then warmed toroom temperature for 10 minutes. After re-cooling to 0° C.,di-tert-butyl dicarbonate (16.1 g, 0.0585 mol) in tetrahydrofuran (100ml) was added dropwise and the mixture was stirred for 30 minutes at 0°C. After a further 2 h at room temperature the reaction was quenched bydropwise addition of saturated sodium bicarbonate (40 ml) and pouredinto water (11). The solution was extracted with ethyl acetate (3×200ml) and the combined extract washed with brine, dried (MgSO₄) andevaporated in vacuo. The residual solid was triturated with petroleumether, removed by filtration and dried in vacuo. The title compound (4.3g, 60%) was obtained as an off-white solid. M.p. 124-127° C.

3-[N-(N′,N″-Di-tert-butoxycarbonyl)-2-guanidinoethyl]-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(3)

1-Methyl-2-chloropyridinium iodide (0.77 g, 3.03 mmol) inN,N-dimethylformamide (4 ml) was added dropwise to a stirred mixture ofN,N′-di-(tert-butoxycarbonyl)thiourea (0.84 g, 3.03 mmol),3-(2-aminoethyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(2) (1.1 g, 2.53 mmol) and triethylamine (0.56 g, 5.57 mmol) inN,N-dimethylformamide (8 ml). After 18 h the reaction was quenched byaddition of water (40 ml) and extracted with ethyl acetate (3×25 ml).The combined extract was washed with brine, dried (MgSO₄) and evaporatedin vacuo. The residual oil was purified by column chromatography onsilica gel (petroleum ether/ethyl acetate, 2:1) to obtain the titlecompound (1.05 g, 61%) as a yellow foam.

M/z [ES]+ 677.

9,10-Dimethoxy-3-[2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

Trifluoroacetic acid (0.35 g, 3.1 mmol was added to a stirred solutionof 3 (0.95 g, 1.4 mmol) in dichloromethane (5 ml) at room temperature.After 3 h further trifluroacetic acid (0.35 g) was added and the mixturewas stirred for an additional 16 h. The solvent was then evaporated invacuo and the residue was treated with dichloromethane (20 ml) andbasified to pH 10 with saturated sodium bicarbonate. The organic phasewas separated, dried (MgSO₄) and evaporated in vacuo. The residual oilwas purified by column chromatography on silica gel (CH₂Cl₂/MeOH,98:2→90:10) to obtain the title compound, after trituration with diethylether, as an off-white solid, 0.27 g, 40%.

M.p.: 226-228° C. M/z: C₂₆H₃₂N₆O₃ requires M = 476, found m/z [ES]+ =477 HPLC: Area (%) 98.73 Column ODS LUNA 3uC18(2) (100 × 4.6 mm) MP 0.1%CF₃CO₂H/CH₃CN (gradient 90% aq → 25% aq over 25 min) RT (min) 11.413 FR(ml/min) 0.8 Detection 250 nm

¹H NMR (250 MHz, CDCl₃, 70° C.): δ 2.03 (6H, s, 2×CH₃), 2.26 (3H, s,CH₃), 2.95 (2H, t, CH₃), 3.57 (2H, m, CH₂), 3.67 (3H, s, OCH₃), 3.85(3H, s, OCH₃), 3.98 (2H, t, CH₂), 4.33 (2H, t, CH₂), 5.40 (1H, s, C═CH),6.73 (1H, s, ArH), 6.91 (2H, s, 2×ArH), 6.98 (1H, s, ArH), 7.25 (2H, brs, NH₂), 7.73 (1H, m, NH).

The ¹H NMR was run at 70° C. to obtain better resolution because some ofthe signals were poorly resolved at room temperature.

EXAMPLE 89,10-Dimethoxy-3-[N-(N′-nitro)-2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

2-Methyl-1-nitro-2-isothiourea (1)

S-Methylisothiouronium sulphate (3.0 g, 10.8 mmol) was added in portionsto a stirred mixture of fuming nitric acid (3 ml) and concentratedsulphuric acid (9 ml) at −10 to +5° C. After stirring for a further 30min at 5° C. the solution was poured onto ice (120 g) with stirring. Thewhite solid was removed by filtration, washed with water and dried invacuo to obtain 2-methyl-1-nitro-2-isothiourea (2.0 g, 69%).

9,10-Dimethoxy-3-[N-(N′-nitro)-2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

2-Methyl-1-nitro-2-isothiourea (0.405 g, 3.0 mmol) was added to astirred solution of3-(2-aminoethyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(1.29 g, 3.0 mmol) in ethanol (12 ml) and heated to 70° C. for 30 min.The solvent was then evaporated in vacuo and the residue was purified bycolumn chromatography on silica gel [CH₂Cl₂/MeOH, 97:3)] to obtain thetitle compound as a pale yellow solid (0.76 g, 49%).

M.p.: 253-256° C. M/z: C₂₆H₃₁N₇O₅ requires M = 521, found m/z[ES]+ = 522HPLC: Area (%) 99.44 Column ODS LUNA 3uC18(2) (100 × 4.6 mm) MP 0.1%CF₃CO₂H/CH₃CN (gradient 90% aq → 25% aq over 25 min) RT (min) 16.842 FR(ml/min) 1.0 Detection 250 nm

¹H NMR (250 MHz, d₆-DMSO, 70° C.): δ 2.02 (6H, s, 2×CH₃), 2.25 (3H, s,CH₃), 2.94 (2H, t, CH₂), 3.63 (2H, m, CH₂), 3.66 (3H, s, OCH₃), 3.84(3H, s, OCH₃), 3.96 (2H, t, CH₂), 4.37 (2H, t, CH₂), 5.38 (1H, s, C═CH),6.72 (1H, s, ArH), 6.88 (21H, s, 2×ArH), 6.96 (1H, s, ArH), 7.89 (1H, brs, NH).

The ¹H NMR was run at 70° C. to obtain better resolution because some ofthe signals were poorly resolved at room temperature.

EXAMPLE 93-[N-(N′-Cyclohexylcarbamoyl)-2-aminoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

Cyclohexylisocyanate (0.38 g, 3.04 mmol) in toluene (2 ml) was addeddropwise to a stirred solution of3-(2-aminoethyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(1.2 g, 2.76 mmol) in toluene (8 ml) at room temperature, undernitrogen. After stirring for 16 h the solution was evaporated in vacuoand the residue was purified by column chromatography on silica gel[dichloromethane/methanol (97:3)]. The product was triturated with etherto obtain the title compound (0.61 g, 40%) as a pale yellow solid.

M.p.: 120-122° C. M/z: C₃₂H₄₁N₅O₄ requires M = 559, found m/z [ES+] =560 HPLC: Area (%) 98.59 Column ODS LUNA 3uC18(2) MP 0.1 M NH₄OAc/CH₃CN(40/60) FR (ml/min) 9.145 RT (min) 0.7 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 0.7-1.8 (11H, m, cyclohexyl), 2.05 (6H, s,2×CH₃), 2.27 (3H, s, CH₃), 2.93 (2H, t, CH₂), 3.49 (2H, m, CH₂), 3.78(3H, s, OCH₃), 3.91 (3H, s, OCH₃), 4.05 (2H, t, CH₃), 4.37 (2H, t, CH₂),5.49 (1H, s, C═CH), 5.80 (1H, br s, NH), 6.69 (1H, s, ArH), 6.70 (1H, s,ArH), 6.90 (2H, s, 2×ArH).

EXAMPLE 103-(N-Carbamoyl-2-aminoethyl)-9,10-dimethoxy-2-(2-methylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

9,10-Dimethoxy-2-(2-methylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(1)

2-Methylaniline (5.44 ml, 51 mmol) and2-chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one(5 g, 17 mmol) were suspended in propan-2-ol (400 ml) and heated atreflux, under nitrogen, for 24 h. After cooling to room temperature, thesolution was concentrated in vacuo and the residue purified by flashcolumn chromatography [dichloromethane/methanol (98:2-96:4)] to affordthe title compound (6.2 g, quantitative yield) as a yellow/orange solid.

9,10-Dimethoxy-2-(2-methylphenylimino)-3-(N-phthalimidoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(2)

A mixture of isoquinoline 1 (6.2 g, 17 mmol), potassium carbonate (21.1g, 153 mmol), sodium iodide (15.3 g, 102 mmol) andN-(2-bromoethyl)phthalimide (25.9 g, 102 mmol) in 2-butanone (170 ml)was stirred at reflux, under nitrogen, for 7 days. After cooling to roomtemperature, the mixture was filtered and the residue washed withmethanol (150 ml). The filtrate was concentrated in vacuo and theresultant residue treated with methanol (100 ml) and the solid filteredoff and washed with methanol. This solid was washed with ether to givethe title compound (2.67 g, 30%) as a pale yellow solid

3-(2-Aminoethyl-9,10-dimethoxy-2-(2-methylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(3)

A mixture of phthalimide 2 (2.66 g, 4.96 mmol) and hydrazine monohydrate(1.24 g, 24.8 mmol) in chloroform (35 ml) and ethanol (60 ml) wasstirred at room temperature, under nitrogen, for 18 h. Additionalhydrazine hydrate (0.25 g, 5 mmol) was added and the mixture stirred fora further 5 h. After cooling to 0° C. in an ice/water bath, the solidwas removed by filtration, the residue washed with a little coldchloroform and the filtrate concentrated in vacuo. This residue wastaken up in dichloromethane and the insoluble material removed byfiltration. The filtrate was dried (MgSO₄), filtered and concentrated invacuo to afford the title compound (2.05 g, quantitative yield) as ayellow foam.

3-(N-Carbamoyl-2-aminoethyl-9,10-dimethoxy-2-(2-methylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

Sodium cyanate (0.64 g, 9.8 mmol) in water (13 ml) was added dropwise toa stirred solution of amine 3 (2 g, 4.9 mmol) in water (63 ml) and 1MHCl (9.8 ml) at 80° C. After stirring for 3 h at 80° C. the mixture wascooled and basified with 2M NaOH. The mixture was extracted with CH₂Cl₂until no more product remained in the organic phase. The organic phaseswere combined, dried (MgSO₄), filtered and concentrated in vacuo. Theresidue was purified by column chromatography [dichloromethane/methanol(97:3)] and the product triturated with ether to afford the titlecompound (1.0 g, 45%) as a pale yellow solid.

M.p.: 236-238° C. m/z: C₂₄H₂₇N₅O₄ requires M = 449, found (M + 1) = 450HPLC: Area 100% Column ODS LUNA (150 4.6 mm) MP pH3 KH₂PO₄/CH₃CNgradient 90% aq going to 50% over 25 mins FR 1.0 ml min⁻¹ RT 14.284 minDetection 250 nm

¹H NMR (300 MHz; d₆-DMSO): δ 2.08 (3H, s, CH₃), 2.89 (2H, t, CH₂), 3.60(3H, s, OMe), 3.79 (3H, s, OMe), 3.91 (2H, t, CH₂), 4.14 (2H, t, CH₂),5.44 (2H, br s, NH₂), 5.64 (1H, S, vinylic H), 6.07 (1H, t, NH), 6.71(1H, s, ArH), 6.73 (1H, d, ArH), 6.92 (1H, t, ArH), 6.95 (1H, s, ArH),7.13 (1H, t, ArH), 7.19 (1H, d, ArH).

NB. The proton NMR spectrum above does not show the chemical shift forone CH₂ group as it is believed that this signal is obscured by thewater signal present in the d₆-DMSO at 3.31-3.35.

EXAMPLE 113-(N-Carbamoyl-2-aminoethyl)-2-(2,6-diisopropylphenylimino)-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

2-(2,6-Diisopropylphenylimino)-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-pyrimido-[6,1-a]isoquinolin-4-one(1)

2,6-Diisopropylaniline (9.62 ml, 51 mmol) and2-chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one(5 g, 17 mmol) were suspended in propan-2-ol (400 ml) and heated atreflux, under nitrogen, for 4 days. After cooling to room temperature,the solution was concentrated in vacuo and the residue purified by flashcolumn chromatography [dichloromethane/methanol (98:2-96:4)] to affordthe title compound (5.8 g, 79%) as a yellow/orange solid.

2-(2,6-Diisopropylphenylimino)-9,10-dimethoxy-3-(N-phthalimidoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(2)

A mixture of isoquinoline 1 (5.8 g, 13.4 mmol), potassium carbonate(16.7 g, 121 mmol), sodium iodide (12.1 g, 80 mmol) andN-(2-bromoethyl)phthalimide (25.9 g, 80 mmol) in 2-butanone (150 ml) wasstirred at reflux, under nitrogen, for 5½ days. After cooling to roomtemperature, the mixture was filtered and the residue washed withmethanol (150 ml). The filtrate was concentrated in vacuo and theresultant residue treated with methanol (100 ml) and the solid filteredoff and washed thoroughly with methanol. The resultant solid was driedto give the title compound (4.9 g, 60%) as a pale yellow solid.

3-(2-Aminoethyl)-2-(2,6-diisopropylphenylimino)-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(3)

A mixture of phthalimide 2 (4.9 g, 8.08 mmol) and hydrazine monohydrate(2.01 g, 40.4 mmol) in chloroform (70 ml) and ethanol (105 ml) wasstirred at room temperature, under nitrogen, for 18 h. Additionalhydrazine hydrate (0.5 g, 10 mmol) was added and the mixture stirred fora further 3 h. After cooling to 0° C. in an ice/water bath, the solidwas removed by filtration, the residue washed with a little coldchloroform and the filtrate concentrated in vacuo. This residue wastaken up in dichloromethane and the insoluble material removed byfiltration. The filtrate was dried (MgSO₄), filtered and concentrated invacuo to afford the title compound (3.24 g, 84%) as a yellow foam.

3-(N-Carbamoyl-2-aminoethyl)-2-(2,6-diisopropylphenylimino)-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

Sodium cyanate (0.87 g, 13.4 mmol) in water (20 ml) was added dropwiseto a stirred solution of amine 3 (3.2 g, 6.7 mmol) in water (100 ml) and1M HCl (13.4 ml) at 80° C. After stirring for 4 h at 80° C. the mixturewas cooled and basified with 2M NaOH. The mixture was extracted withCH₂Cl₂ until no more product remained in the organic phase. The organicphases were combined, dried (MgSO₄), filtered and concentrated in vacuo.The residue was purified by column chromatography[dichloromethane/methanol (97:3)] and the isolated product taken up in amixture of dichloromethane and ether which on concentration afforded thetitle compound (0.6 g, 17%) as a pale yellow foam.

M.p.: 213-215° C. m/z: C₂₉H₃₇N₅O₄ requires M = 519, found (M ÷ 1) = 520HPLC: Area 97.59% Column ODS LUNA (150 4.6 mm) MP pH3 KH₂PO₄/CH₃CNgradient 90% aq going to 50% over 25 mins FR 1.0 mlmin⁻¹ RT 10.723 minDetection 250 nm

¹H NMR (300 MHz; DMSO): 1.07 (12H,dd, 4 CH₃), 2.82-2.94 (4H, m, CH₂ and2 CH(CH₃)₂, 3.55 (3H, s, OMe), 3.78 (3H, s, OMe), 3.91 (2H, t, CH₂),4.17 (2H, t, CH₂), 5.32 (1H, s, vinylic H), 5.45 (2H, br s, NH₂), 6.13(1H, t, NH), 6.56 (1H, s, ArH), 6.95 (1H, s, ArH), 7.00 (1H, m, ArH),7.10 (1H, s, ArH), 7.12 (1H, br s, ArH).

NB. The proton NMR spectrum above does not show the chemical shift forone CH₂ group as it is believed that this signal is obscured by thewater signal present in the d₆-DMSO at 3.30-3.33.

EXAMPLE 123-(N-Carbamoyl-4-aminobutyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

9,10-Dimethoxy-3-(4-N-phthalimidobutyl)-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(1)

A mixture of9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(4.0 g, 10.2 mmol), N-(4-bromobutyl)phthalimide, (8.6 g, 30.6 mmol),potassium carbonate (12.7 g, 91.8 mmol) and sodium iodide (4.6 g, 30.6mmol) in 2-butanone (100 ml) was stirred at reflux, under nitrogen for 4days. After cooling to room temperature, the solid was removed byfiltration and the filtrate was evaporated in vacuo. The residual solidwas purified by column chromatography on silica gel [petroleumether/ethyl acetate (2:1)-(1:1)]. The title compound (1.45 g, 24%) wasobtained as a yellow solid.

3-(4-Aminobutyl-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(2)

A solution of 1 (1.4 g, 2.36 mmol) in ethanol (30 ml) and chloroform (20ml) was treated with hydrazine hydrate (0.42 g, 7.09 mmol) and stirredat room temperature, under nitrogen. After 18 h further hydrazinehydrate (0.42 g) was added and stirred for an additional 5 h. Thereaction mixture was then cooled to 0° C. and the solid was removed byfiltration. The filtrate was dried (MgSO₄) and evaporated in vacuo toobtain the title compound (1.0 g, 92%) as a yellow solid.

3-(N-Carbamoyl-4-aminobutyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

Sodium cyanate (0.28 g, 4.32 mmol) in water (6 ml) was added dropwise toa stirred solution of 2 (1.0 g, 2.16 mmol) in water (30 ml) and 1N HCl(4.3 ml) at 80° C. After 2 h at 80° C. the mixture was cooled andextracted with dichloromethane (3×20 ml) and the extract dried (MgSO₄)and evaporated in vacuo. The residue was purified by columnchromatography on silica gel [dichloromethane/methanol (97:3)] to obtainthe title compound (0.70 g, 64%) as a yellow solid.

M.p: 234-235° C. M/z C₂₈H₃₅N₅O₄ requires M = 505, found m/z [ES+] = 506HPLC: Area (%) 98.94 Column ODS LUNA 3uC18(2) (100 × 4.6 mm) MP 0.02MKH₂PO₄/CH₃CN (gradient 90% aq → 50% aq over 25 min) RT (min) 17.201 FR(ml/min)  1.0 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 1.64 (2H, m, CH₂), 1.90 (2H, m, CH₂), 2.07(6H, s, 2×CH₃), 2.26 (3H, s, CH₃), 2.92 (2H, t, CH₂), 3.30 (2H, m, CH₂),3.73 (3H, s, OCH₃), 3.88 (3H, s, OCH₃), 4.06 (2H, t, CH₂), 4.27 (4H, m,CH₂+NH₂), 5.44 (1H, s, C═CH), 6.65 (1H, s, ArH), 6.67 (1H, s, ArH), 6.89(2H, s, 2×ArH).

EXAMPLE 133-[N-(N′-Cyano-N″-methyl)-2-guanidinoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

3-[N-(N′-Cyano-S-methyl)-2-isothioureidoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(1)

Dimethyl N-cyanodithioiminocarbonate (7.45 g, 46.1 mmol) was added to asolution of3-(2-aminoethyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one(2.0 g, 4.61 mmol) in toluene (50 ml) and stirred at 90° C. undernitrogen. After 2 h the solvent was evaporated in vacuo and the residuewas purified by column chromatography on silica gel[dichloromethane/methanol (100:0)-(95:5)]. The title compound (2.30 g,94%) was obtained as a yellow solid.

3-[N-(N′-Cyano-N″-methyl)-2-guanidinoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one

A solution of 1 (2.0 g, 3.76 mmol) in dichloromethane (30 ml) wastreated with 2M methylamine/THF (9.4 ml, 18.8 mmol) and stirred atreflux, under nitrogen. After 16 h additional 2M methylamine/THF (9.4ml) was added, followed by a further two portions of 2M methylamine/THF(9.4 ml) at 2 h intervals. After 24 h at reflux the reaction was cooledand evaporated in vacuo. The residue was purified by columnchromatography on silica gel [dichloromethane/methanol (98:2)] toobtain, after trituration with ether, the title compound (1.20 g, 62%)as a pale yellow solid.

M.p.: 223-224° C. M/z: C₂₈H₃₃N₇O₃ requires M = 515, found m/z [ES+] =516 HPLC: Area (%) 100 Column ODS LUNA 3uC18(2) (100 × 4.6 mm) MP 0.02MKH₂PO₄/CH₃CN (gradient 90% aq → 50% aq over 25 min) RT (min)  17.838 FR(ml/min)  1.0 Detection 250 nm

¹H NMR (300 MHz, CDCl₃): δ 1.99 (6H, s, 2×CH₃), 2.22 (3H, s, CH₃), 2.43(3H, d, NHCH₃), 2.86 (2H, t, CH₂), 3.52 (2H, m, CH₂), 3.69 (3H, s,OCH₃), 3.83 (3H, s, OCH₃), 4.02 (2H, t, CH₂), 4.28 (2H, m, CH₂), 5.39(1H, s, C═CH), 6.61 (1H, s, ArH), 6.63 (1H, s, ArH), 6.82 (2H, s,2×ArH).

The pharmacological utility of the compounds of the present inventionhas been demonstrated in studies using compounds previously synthesisedfrom the above Examples. The results below serve to illustrate thegeneric application of the compounds of the present invention.

EXAMPLE A Efficacy of a Compound of the Invention AgainstElectrical-Induced Contraction of Guinea-Pig Isolated Trachea

The efficacy of the compounds of Example 1, 8, 9, 10, 11 and 13 weretested against electrical-induced contraction of guinea-pig isolatedtrachea. The results demonstrate that the compounds of the presentinvention inhibit the contractile responses with a long duration ofaction.

Method

Superfusion of guinea-pig tracheal rings was performed according to apreviously described method (Coleman et al. 1996; PulmonaryPharmacology, 9, 107-117). Briefly, guinea-pig tracheal preparationswere cut into rings then opened by sectioning the ring opposite thesmooth muscle and suspended between two platinum electrodes under 1 gtension. The tissues were superfased at a rate of 3.25 ml/min withKrebs-Henseleit solution at 37° C. containing the cyclooxygenaseinhibitor, indomethacin (5 μM) and bubbled with 95% O₂ and 5% CO₂.Tracheal preparations were allowed to equilibrate for 40 min beforecommencement of electrical stimulation delivered as a 10 s train ofsquare wave pulses at 3 Hz, 0.1 ms duration and 20V (approx. 400 mAmps)generated every 100 sec by means of physiological squarewave-stimulator.

The compound of Example 1 was dissolved in DMSO containing Tween 80(10%) and distilled water (0.01M), which were then added to the organbath to give a final concentration of 10 μM. The other compounds wereprepared in DMSO and diluted in Krebs-Henseleit solution which yielded afinal superfusion concentration of 0.05% DMSO, and superfused at a rateof 0.3 ml/min; contractile responses to electrical field stimulation wasrecorded on a Macintosh computer using MacLab software.

Results

The vehicle, DMSO, failed to significantly inhibit the contractileresponse to electrical field stimulation (FIG. 2). The results for thecompounds are shown in Figures: 3 for compound of Example 1; 4 forcompound of Example 9; 5 for compound of Example 10; 6 for compound ofExample 11; 7 for compound of Example 13; and 8 for compound of Example8.

The compounds caused complete inhibition of the contractile response toelectrical field stimulation and the effect was maintained for more than2-4 hours.

EXAMPLE B Efficacy of a Compound of the Invention Against Proliferationof Human Mononuclear Cells Stimulated by PHA

The effect of the compound of Example 1,9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,against proliferation of human mononuclear cells stimulated by PHA wasinvestigated. Proliferation was significantly inhibited by the compound,indicating that it possesses anti-inflammatory activity. The resultbelow serves to illustrate the generic application of the novelcompounds of the present invention.

Method

Normal healthy volunteers underwent phlebotomy and 25 ml of blood wascollected. Mononuclear cells were separated and purified according tothe method of Banner et al. (Banner et al. Br. J. Pharmacol. 1163169-3174 (1995)). Human peripheral mononuclear cells (100,000 per well)were stimulated for 24 h with phytohaemagglutinin (PHA, 2 μg/ml) in theabsence or presence of the compound of Example 1 (0.001-100 μM) at 37°C. in a 95% air, 5% CO₂ atmosphere. Twenty four hours later,[³H]-thymidine (0.1 μCi) was added to each well and the cells wereincubated for a further 24 h period. Cells were then harvested ontoglass fibre filters using a cell harvester (ICN Flow, Buckinghamshire)and counted in a scintillation counter.

Results

The compound under test caused a concentration dependent inhibition ofthe proliferation of human mononuclear cells stimulated with PHA (numberof experiments is 6; FIG. 8). The IC₅₀ values and 95% confidence limitsfor these compounds are indicated in Table 1. The result is also shownin the graph of FIG. 8.

TABLE 1 IC₅₀ value for the compound of Example 1 against proliferationof human mononuclear cells stimulated with PHA Compound from IC₅₀ nExample 1 0.46 μM (0.239-0.897) 6

EXAMPLE C Inhibition of Phosphodiesterase (PDE) Type 3 and 4 Isozymes

The compound of Example 1,9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,has been shown to be a potent inhibitor of phosphodiesterase (PDE) type3 and 4 isozymes. The IC₅₀ values are shown below.

PDE4 (nM) PDE3 (nM) (human (human platelet) neutrophil) Compound ofExample 1    0.43 1479 Rolipram ND 6412 Cilostamide 89 ND

Rolipram is a known PDE 4 inhibitor and cilostamide is a known PDE3inhibitor ND-Not determined

EXAMPLE D Effect of the Compound of Example1,9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,on LPS Induced TNF-α Release From Human Monocytes

IC50 (nM) Compound of Example 1   7.5 n = 6 CDP 840 (PDE4 inhibitor)  92n = 6 Siguazodan (PDE3 inhibitor) >100 μM

EXAMPLE E In Vivo Tests

1. The compound of Example 1,9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one,was tested in a model of histamine induced bronchospasm. Consciousguinea-pigs were exposed to dry powder (micronised) compound. The drugwas blended with lactose so that the final concentration was 0.25, 2.5and 25%. At various times after exposure to drug the animals wereanaesthetised and challenged with varying doses of histamine. Totalairway resistance and mean arterial blood pressure were recorded.

Exposure to dry powder (2.5 and 25%) provided significant protectionagainst histamine induced bronchospasm over a 5.5 hour period andreduced mean blood pressure over this period.

2. Intravenous administration of the compound (1 to 100 μg/kg) tourethane anaesthetised guinea-pigs produced a dose dependant reductionin mean arterial blood pressure. The compound was dissolved in DMSO thendiluted with saline (there was evidence that the compound had come outof solution).

3. In a model of antigen induced eosinophilia in the ovalbuminsensitised guinea-pig, the compound (10 mg/kg) administered orally 1hour prior to antigen challenge, significantly inhibited the recruitmentof eosinophils to the lungs following antigen challenge (aerosol) insensitised guinea-pigs. Exposure to dry powder (25%), 1.5 hours prior toantigen challenge, significantly inhibited recruitment of eosinophils tothe lungs (measurements were made 24 hours after challenge).

Further experiments were carried out to characterise the duration ofaction in this model. Compound (25%) administration 5.5 hours beforeantigen challenge failed to significantly inhibit eosinophil recruitmentto the lungs.

EXAMPLE F Taste of Compounds

For pharmaceutical compounds which are administered orally, taste is avery important factor in ensuring patient compliance. Unexpectedly, thecompounds of the present invention are substantially tasteless. They aretherefore particularly suitable for oral administration, for example asdry powder to be inhaled.

Method

Small amounts of the compound of Example 1, trequinsin(9,10-dimethoxy-3-methyl-2-mesitylimino-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one)and desmethyl trequinsin(9,10-dimethoxy-2-mesitylimino-2,3,6,7-tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one)were placed on the tip of the tongue of an informed, healthy malevolunteer and the taste of each compound was assessed.

Results

The results, displayed in Table 3 below, show that the compound ofExample 1 has significantly improved taste compared wth trequinsin ordesmethyl trequinsin.

Compound (from) Taste¹ Example 1 *** Trequinsin * Desmethyl trequinsin *¹Scale: * Very bitter ** Bitter *** Tasteless

1. A method to treat asthma or to cause bronchial dilation in a mammalin need thereof, which method comprises administering to said mammal aneffective, non-toxic amount of a compound of formula I:

wherein each of R¹ and R² independently represents a C₁₋₆ alkyl or C₂₋₇acyl group; R⁵ represents a hydrogen atom or a C₁₋₃ alkyl, C₂₋₃ alkenylor C₂₋₃ alkynyl group; R⁶ represents a hydrogen atom or a C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, amino, C₁₋₆ alkylamino, di(C₁₋₆) alkylaminoor C₂₋₇ acylamino group; each of R⁷ and R⁸ independently represents ahydrogen or halogen atom or a hydroxy, trifluoromethyl, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₂₋₇ acyl, C₁₋₆ alkylthio, C₁₋₆ alkoxy, C₃₋₆cycloalkyl; and R⁹ represents a hydrogen or halogen atom or a hydroxy,trifluoromethyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₇ acyl, C₁₋₆alkylthio, C₁₋₆ alkoxy or C₃₋₆ cycloalkyl group; X represents a groupCR³R⁴, wherein each of R³ and R⁴ independently represents a hydrogenatom or a C₁₋₃ alkyl group; each of R¹⁰ and R¹¹ independently representsa hydrogen atom, a C₁₋₃ alkyl, C₃₋₆ cycloalkyl or phenyl group; Yrepresents an oxygen atom or a group CHNO₂, NCN, NH or NNO₂; n is anintegerfrom 2 to 4; or a salt thereof.
 2. A method for the treatment ofchronic obstructive pulmonary disease (COPD) in a mammal, which methodcomprises administering to said mammal an effective, non-toxic amount ofa compound of formula I:

wherein each of R¹ and R² independently represents a C₁₋₆ alkyl or C₂₋₇acyl group; R⁵ represents a hydrogen atom or a C₁₋₃ alkyl, C₂₋₃ alkenylor C₂₋₃ alkynyl group; R⁶ represents a hydrogen atom or a C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, amino, C₁₋₆ alkylamino, di(C₁₋₆) alkylaminoor C₂₋₇ acylamino group; each of R⁷ and R⁸ independently represents ahydrogen or halogen atom or a hydroxy, trifluoromethyl, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₂₋₇ acyl, C₁₋₆ atkylthio, C₁₋₆ alkoxy, C₃₋₆cycloalkyl; and R⁹ represents a hydrogen or halogen atom or a hydroxy,trifluoromethyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₇ acyl, C₁₋₆alkylthio, C₁₋₆ alkoxy or C₃₋₆ cycloalkyl group; X represents a groupCR³R⁴, wherein each of R³ and R⁴ independently represents a hydrogenatom or a C₁₋₃ alkyl group; each of R¹⁰ and R¹¹ independently representsa hydrogen atom, a C₁₋₃ alkyl, C₃₋₆ cycloalkyl or phenyl group; Yrepresents an oxygen atom or a group CHNO₂, NCN, NH or NNO₂; n is aninteger from 2 to 4; or a salt thereof.
 3. A method as claimed in anyone of claims 1 or 2, wherein independently or in any compatiblecombination: each of R¹ and R² independently represent a C₁₋₆ alkyl;each of R³ and R⁴ represents a hydrogen atom; R⁵ represents a hydrogenatom; R⁶ represents a hydrogen atom; each of R⁷ and R⁸ independentlyrepresent a C₁₋₆ alkyl; R⁹ represents a halogen atom or a methyl oracetyl group; Y represents an oxygen atom or a group CHNO₂; and n is 2.4. A method as claimed in any one of claims 1 or 2, wherein the compoundis administered by aerosol.
 5. A method as claimed in any one of claims1 or 2, wherein the mammal is a human.
 6. A method as claimed in any oneof claims 1 or 2, wherein each of R¹ and R² represents a C₁₋₄ alkylgroup; and each of R⁷ and R⁸ represents a methyl, ethyl or isopropylgroup.
 7. A method as claimed in any one of claims 1 or 2, wherein thecompound of formula I is selected from the group consisting of:9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-(N-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-(N′-isopropylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-methyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′-isopropyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-[1-(N′,N′-dimethyl-2-nitroethenamine)]-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;9,10-Dimethoxy-2-(2,4,6-trimethylphenylimino)-3-[N-(N′-phenylcarbamoyl)-2-aminoethyl]-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-2-one;9,10-Dimethoxy-3-[2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;9,10-Dimethoxy-3-[N-(N′-nitro)-2-guanidinoethyl]-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;3-[N-(N′-Cyclohexylcarbamoyl)-2-aminoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;3-(N-Carbamoyl-2-aminoethyl)-9,10-dimethoxy-2-(2-methylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;3-(N-Carbamoyl-2-aminoethyl)-2-(2,6-diisopropylphenylimino)-9,10-dimethoxy-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;3-(N-Carbamoyl-4-aminobutyl)-9,10-dimethoxy-2-(2,4,6-trimethylphenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one;and3-[N-(N′-Cyano-N″-methyl)-2-guanidinoethyl]-9,10-dimethoxy-2-(2,4,6-trimethyl-phenylimino)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one.8. A method of treating a respiratory disorder in a mammal, comprisingadministering to a subject an effective, non-toxic amount of a compoundof formula I:

wherein each of R¹ and R² independently represents a C₁₋₆ alkyl or C₂₋₇acyl group; R⁵ represents a hydrogen atom or a C₁₋₃ alkyl, C₂₋₃ alkenylor C₂₋₃ alkynyl group; R⁶ represents a hydrogen atom or a C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, amino, C₁₋₆ alkylamino, di(C₁₋₆) alkylaminoor C₂₋₇ acylamino group; each of R⁷ and R⁸ independently represents ahydrogen or halogen atom or a hydroxy, trifluoromethyl, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₂₋₇ acyl, C₁₋₆ alkylthio, C₁₋₆ alkoxy, C₃₋₆cycloalkyl; and R⁹ represents a hydrogen or halogen atom or a hydroxy,trifluoromethyl, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₇ acyl, C₁₋₆alkylthio, C₁₋₆ alkoxy or C₃₋₆ cycloalkyl group; X represents a groupCR³R⁴, wherein each of R³ and R⁴ independently represents a hydrogenatom or a C₁₋₃ alkyl group; each of R¹⁰ and R¹¹ independently representsa hydrogen atom, a C₁₋₃ alkyl, C₃₋₆ cycloalkyl or phenyl group; Yrepresents an oxygen atom or a group CHNO₂, NON, NH or NNO₂; n is aninteger from 2 to 4; or a salt thereof, and wherein the respiratorydisorder is selected from the group consisting of allergic asthma, hayfever, allergic rhinitis.
 9. The method of claim 8, wherein therespiratory disorder is selected from the group consisting of hay feverand allergic rhinitis.